PROJECT SUMMARY Over 100 million people worldwide drink water and eat food containing unsafe levels of inorganic arsenic (iAs). iAs is a potent human carcinogen. Chronic iAs exposure is also associated with increased risk of cardiometabolic, neurological, and respiratory diseases. In addition, gestational and early-life exposures to iAs are linked to low birth weight, infant mortality, and developmental neurotoxicity in children. In spite of extensive research over the past 20+ years, the mechanisms underlying the adverse effects of iAs exposure remain largely unclear, partly due to the lack of an adequate animal model. Many health effects reported in humans have not been reproduced in the laboratory because iAs metabolism in laboratory animals differs from that in humans. Strong evidence from both population and laboratory studies suggests that the differences in the structure and function of human arsenic methyltransferase (AS3MT) and mouse As3mt are responsible for the differences in iAs metabolism between humans and mice which, in turn, determine the differences in the susceptibility of these species to the adverse effects of iAs exposure. The ultimate goal of this project is to generate a mouse model in which the metabolism and adverse effects of iAs exposure described in humans can be reproduced and studied. We have developed vector systems for manipulation of the AS3MT/As3mt locus in both mouse and human pluripotent stem cells. We have used these systems to replace the As3mt locus in mouse embryonic stem cells (ES) with the syntenic segment of human DNA using CRISPR/cas9 augmented homologous recombination. The specific aims of this project are: 1. To compare the metabolism of iAs in mouse ES cells expressing human AS3MT and in primary cells (endodermal cells and hepatocytes) derived from these ES cells with iAs metabolism in human ES cells and differentiated cells. 2. To generate and characterize a mouse strain expressing human AS3MT. Mouse ES cells expressing AS3MT will be injected into blastocysts to generate mice expressing human AS3MT in the place of the mouse gene. To characterize the humanized mouse strain, we will compare AS3MT expression and iAs metabolism in the humanized mice with those described for humans. We will also characterize metabolic phenotype of the humanized mice exposed to iAs in drinking water at the levels that were associated with adverse metabolic phenotypes in human studies. Creation of a humanized mouse model represents a major innovation that will allow us to reproduce and study in the laboratory the adverse effects of iAs exposure found in population studies. This project has a potential to move forward the entire field of As toxicology and contribute significantly to our understanding of the disease associated with iAs exposure, providing key information for efficient treatment or prevention of this disease.